how it works
The CyQUANT® Direct assay is based on a cell-permeant DNA-binding dye in combination with a masking dye. The masking dye blocks staining of dead cells and cells with compromised cell membranes so that only viable cells fluoresce. Because DNA content is highly regulated, cell number estimates using the CyQUANT® Direct assay are very accurate.

CyQUANT® Direct assay protocol. The CyQUANT® Direct assay is designed for use with multi-well plates (96-, 384-, or 1,536-well formats), making it ideal for high-throughput screening applications. The reagent is added directly to cells in complete medium and incubated for 30 to 60 min. Samples are read from the bottom on standard fluorescence plate readers set for green fluorescence (e.g., fluo-4 and GFP).

how they work
HCS CellMask™ stains label the whole cell, including cytoplasm and nucleus, and are applied to cells immediately after fixation and permeabilization or after antibody labeling. The versatile HCS NuclearMask™ stains measure DNA content in addition to enabling robust cell demarcation of live and formaldehyde-fixed cells.

To learn more about CellMask™ and NuclearMask™ reagents and other tools for high-content imaging and analysis, visit www.invitrogen.com/hcs.

what it is
LIVE/DEAD® cell viability assays are used to differentially stain live and dead cells in a variety of mammalian cell types. These cells can then be fixed with formaldehyde for subsequent analysis by flow cytometry. The LIVE/DEAD® Fixable Yellow dye is excited with a 405 nm violet laser and has an emission maximum of ~570 nm. These spectral properties allow you to transfer your dead-cell discrimination assays to the violet laser, thus freeing up the commonly used 488 nm laser for bright fluorochromes on antibodies directed against challenging antigens.

what it offers

Post-fixation measurement of viability

Accurate intracellular staining

Ideal for multicolor experiments

how it works
LIVE/DEAD® Fixable Dead Cell Stain Kits are based on the reaction of a fluorescent reactive dye with amines on cellular proteins. With viable cells, only cell surface proteins are available to react with the dye, resulting in faint staining. In contrast, the dye can penetrate the interior of dead cells, resulting in at least a 50-fold increase in fluorescence compared to live cells. Because the dye reacts covalently with proteins, the staining pattern is completely preserved following sample fixation with formaldehyde, under conditions that inactivate pathogens.

what they are
Molecular Probes® Organelle Lights™ reagents are prepackaged and ready-to-use fluorescent protein constructs fused with signal peptides for accurate and specific targeting to subcellular compartments and structures. The collection of Organelle Lights™ reagents has recently expanded to include new colors for staining lysosomes, mitochondria, endoplasmic reticulum (ER), and endosomes.

what they offer

Ready-to-use reagents—no potentially harmful cell treatments required

Simple method—optimize transduction conditions only once per cell type

Versatility—multiplex with other stains in live or fixed cells

how they work
Organelle Lights™ reagents consist of suspensions of baculovirus carrying an expression construct with a fluorescent protein fused to either a cellular protein or a localization peptide. Simply add the virus suspension to your cells, incubate overnight, and you’re ready for imaging. Organelle Lights™ reagents can label a broad range of mammalian cell types, including primary and stem cells, without the need for lipids or dye-loading protocols that can perturb cell growth and viability.

Learn more about Organelle Lights™ reagents and other tools based on powerful BacMam technology.

NEW APPLICATIONS

Distinguish Artifacts from True Functional Changes
The use of fluorescent dyes, such as tetramethylrhodamine, methyl or ethyl ester (TMRM or TMRE) to report changes in mitochondrial membrane potential is well established. However, it is often important to determine if an apparent change in the signal from a potentiometric dye is due to a change in mitochondrial function or to an artifact resulting from a change in mitochondrial mass, shape, or movement. For more accurate measurement of mitochondrial membrane potential, these membrane potential–sensitive dyes can be combined with probes for mitochondrial morphology (such as Organelle Lights™ Mitochondria GFP) to measure changes in potential that are independent of mitochondrial mass or movement.

Ratiometric Imaging of Mitochondrial Membrane Potential
Ratiometric imaging of mitochondrial membrane potential can be achieved by first transducing cells with an Organelle Lights™ mitochondria-targeted reagent, and subsequently loading cells with a fluorescent dye that is readily sequestered by active mitochondria. For example, colocalization of green-fluorescent Organelle Lights™ Mito-GFP and red-fluorescent TMRM can be used to generate a pseudoratiometric measurement of mitochondrial membrane potential (see figure). This approach enables the observation of “flickers” in mitochondrial membrane potential that cannot be attributed to movement of mitochondria between focal planes or to a change in mitochondrial mass. A similar approach can be used to monitor mitochondrial calcium with the rhod-2 AM dye or mitochondrial superoxide production with MitoSOX™ Red indicator.

Dynamic imaging of mitochondrial membrane potential. Mitochondria of HeLa cells were labeled with Organelle Lights™ Mitochondria GFP (Cat. no. O36210) (A1), and loaded with 50 nM TMRM (Cat. no. T668) for 10 minutes at 37°C (A2). Colocalization of TMRM and GFP can be clearly seen (A3), confirming the specific accumulation of TMRM in mitochondria. (B) Images were acquired at 5 second intervals for 90 seconds. Polarized mitochondria display both red and green fluorescence; those that have depolarized lose red TMRM fluorescence but retain GFP fluorescence and therefore appear green (B4, and quantified in C). Over time, mitochondrial membrane potential is lost in one mitochondrion (denoted by the arrow in B1) whereas surrounding mitochondria remain polarized. The recovery of membrane potential in this single mitochondrion can be seen in the subsequent image. GFP and TMRM were imaged using standard FITC and TRITC filters, respectively, on a DeltaVision® Core microscope with a 40x lens.

How can we rapidly and reliably assess nucleotide excision repair deficiencies?
Xeroderma pigmentosum (XP), a genetic disorder of the nucleotide excision repair (NER) system, predisposes its sufferers to photosensitivity and UV-induced skin damage. Diagnosis of XP typically entails determining the level of damage-induced, non–S-phase, gap-filling DNA repair activity (termed "unscheduled DNA synthesis", or UDS). While UDS can be sensitively and accurately assayed by monitoring the incorporation of 3H thymidine, this methodology is extremely time- and labor-intensive, and incurs the problems associated with handling radioactive materials. Immunofluorescence assays based on BrdU incorporation are substantially faster, but sensitivity is also greatly reduced as compared to 3H thymidine.

Methods
In this study, Limsirichaikul and colleagues compared these two assays to one based on incorporation of Click-iT® EdU, a reactive thymidine analog that enables fluorescence detection by conjugation to an azide-containing fluorophore ("click" chemistry).

Results
Using UVC-irradiated primary human fibroblasts and detection with Alexa Fluor® 488 azide, the group reported comparable sensitivity with Click-iT® EdU to that observed with conventional 3H thymidine autoradiography. Furthermore, the total time required to perform the EdU assay—about half a day—was dramatically less than that required for the 3H thymidine–based assay and even modestly faster than the BrdU-based assay. The Click-iT® EdU assay was also shown to be compatible with immunostaining and latex-bead labeling, allowing for the incorporation of internal controls that are crucial to rigorous lab testing. The group suggests that Click-iT® EdU–based assays could become the standard tool for the diagnosis of XP.

Free online technical webinars
You are invited to join us for a series of biweekly technical webinars from the comfort of your desk. The webinars will initially focus on imaging-related applications, but we welcome your feedback for additional topics throughout the course of the year. Upcoming topics will be announced each month via email.

Presentations will last approximately 45 minutes, followed by 15 minutes for live Q&A.

Mitochondrial structure and function are key indicators of cellular stress, and mitochondrial defects have been implicated in several neurodegenerative diseases, including Alzheimer’s disease. Recent evidence suggests that the balance between mitochondrial fission and fusion is disrupted in response to several factors implicated in Alzheimer's disease. In addition, factors related to stroke, such as oxidative and nitrosative stress, and calcium dysregulation, can disrupt this balance. All of these effects may be mediated by a key motor protein surrounding mitochondria, dynamin-related protein-1 (Science 324:102 (2009)) or the GTPase mitofusin 2 (Curr Opin Cell Biol 18:453 (2006))
. These findings show that morphological visualization of mitochondria in live or fixed cells can be used as an effective model to understand inducers of these and other neurological pathologies.

New Web Resource for Fluorescence Imaging Accessories
Our new web page dedicated to imaging accessories makes it easier than ever to find imaging tools to help you get the most from your fluorescence imaging experiments—from microscope reference standards to sample preparation systems.

Put Your Flow Cytometer’s Violet Laser to Work
Invitrogen is the recognized leader in providing world-class fluorescent reagents and antibody conjugates designed specifically for use on the violet laser. From immunophenotyping to dead-cell discrimination to cell cycle analysis, our portfolio unlocks the full multicolor potential of violet laser–equipped flow cytometers.

What your colleagues are saying about the Countess™ Automated Cell Counter The Countess™ Automated Cell Counter uses trypan blue staining combined with a sophisticated image analysis algorithm to enable accurate cell and viability counts in just 30 seconds. The algorithm also measures the average size of live, dead, and total cells to give you all the data you need from your cell cultures without using a hemocytometer. Here’s what researchers are saying about how the Countess™ Automated Cell Counter has benefited their research:

“The Countess makes tissue culture work much easier and more productive. I wish I had one years ago!” –John McGrath, Dana-Farber Cancer Institute

“The Countess has worked out well for our lab. The Countess cell counts agree with our manual cell counts, and the Countess is much faster.” –Danielle Krebs, UBC Life Sciences Centre

“We found the Countess very helpful for our migration assays. Instead of spending hours counting cells to get results, we are able to quickly quantify our data! A definite time saver and well worth the cost!” –Holly, University of Rochester

“The Countess saves us hours of time during experimentally intense work days. We also appreciate the consistency of counts even with different users.” –Sarah, University of Illinois